This application relates to recombinant laminin 5 and methods for its use.
Basal laminae (basement membranes) are sheet-like, cell-associated extracellular matrices that play a central role in cell growth, tissue development, and tissue maintenance. They are present in virtually all tissues, and appear in the earliest stages of embryonic development.
Basal laminae are central to a variety of architectural and cell-interactive functions. (See for example, Malinda and Kleinman, Int. J. Biochem. Cell Biol. 28:957-959 (1996); Aumailley and Krieg, J. Invest. Dermatology 106:209-214 (1996)):
1. They serve as architectural supports for tissues, providing adhesive substrates for cells.
2. They create perm-selective barriers between tissue compartments that impede the migration of cells and passively regulate the exchange of macromolecules. These properties are illustrated by the kidney glomerular basement membrane, which functions as an important filtration structure, creating an effective blood-tissue barrier that is not permeable to most proteins and cells.
3. Basal laminae create highly interactive surfaces that can promote cell migration and cell elongation during embryogenesis and wound repair. Following an injury, they provide a surface upon which cells regenerate to restore normal tissue function.
4. Basal laminae present information encoded in their structure to contacting cells that is important for differentiation and tissue maintenance. This information is communicated to the cells through various receptors that include the integrins, dystroglycan, and cell surface proteoglycans. Signaling is dependent not only on the presence of matrix ligands and corresponding receptors that interact with sufficient affinities, but also on such topographical factors as ligand density in a three-dimensional matrix xe2x80x9clandscapexe2x80x9d, and on the ability of basal lamina components to cluster receptors. Because these matrix proteins can be long-lived, basal laminae create a xe2x80x9csurface memoryxe2x80x9d in the basal lamina for resident and transient cells.
The basal lamina is largely composed of laminin and type IV collagen heterotrimers that in turn become organized into complex polymeric structures. To date, six type IV collagen chains and at least twelve laminin chains (and twelve different heterotrimeric laminins) have been identified. These chains possess shared and unique functions and are expressed with specific temporal (developmental) and spatial (tissue-site specific) patterns.
Laminins are a family of heterotrimeric glycoproteins that reside primarily in the basal lamina. They function via binding interactions with neighboring cell receptors, and are important signaling molecules that can strongly influence cellular function. Laminins are important in both maintaining cell/tissue phenotype as well as promoting cell growth and differentiation in tissue repair and development.
Laminins are large, multi-domain proteins, with a common structural organization. The laminin molecule integrates various matrix and cell interactive functions into one molecule.
The laminin molecule is comprised of xcex1-, xcex2-, and xcex3-chains joined together through a coiled-coil domain. Within this structure are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, and membrane-bound receptors. Domains VI, IVb, and IVa form globular structures, and domains V, IIIb, and IIIa (which contain cysteine-rich EGF-like elements) form rod-like structures. (Kamiguchi et al., Ann. Rev. Neurosci. 21:97-125 (1998)) Domains I and II of the three chains participate in the formation of a triple-stranded coiled-coil structure (the long arm).
Table 1 shows the individual chains that each laminin type is composed of:
Four structurally-defined family groups of laminins have been identified. The first group of five identified laminin molecules all share the xcex21 and xcex31 chains, and vary by their xcex1-chain composition (xcex11 to xcex15 chain). The second group of five identified laminin molecules all share the xcex21 and xcex31 chain, and again vary by their xcex1-chain composition. The third group of identified laminin molecules has one identified member, laminin 5, with a chain composition of xcex13xcex23xcex32. The fourth group of identified laminin molecules has one identified member, laminin 12, with the newly identified xcex33 chain (xcex12xcex21xcex33).
Some progress has been made in elucidating the relationship between domain structure and function. (See, for example, Wewer and Engvall, Neuromusc. Disord. 6:409-418 (1996).) The overall sequence similarity among the homologous domains in different chains varies, but it is highest in domain VI (thought to play a key role in laminin polymerization), followed by domains V (possibly involved in protein-protein interactions) and III (entactin/nidogen binding; possible cell adhesion sites), and is lowest in domains I, II (both thought to be involved in intermolecular assembly, and containing possible cell adhesion sites), and G. Not all domains are present in all 3 types of chains. The globular G domain (thought to be involved in cell receptor binding) is present only in the xcex1 chains. Other domains may not be present in all chains within a certain chain type. For example, domain VI is absent from xcex13, xcex14, and xcex32 chains. (Wewer and Engvall, 1996)
As a result of their large size ( greater than 600 kD) and unique structure, the laminin molecules can be resolved in the electron microscope. (Wewer and Engvall, 1996) Typically, laminins appear as cross-shaped molecules in an EM. The three short arms of the cross represent the amino terminal portions of each of the three separate laminin chains (one short arm per chain). The long arm of the cross is composed of the C-terminal parts of the three chains, which together form a coiled coil structure. (Wewer and Engvall, 1996) The long arm ends with the globular G domain.
The coiled-coil domain of the long arm is crucial for assembly of the three chains of laminin. (Yurchenco et al., Proc. Natl. Acad. Sci. 94:10189-10194 (1997)). Disulfide bonds bridge and stabilize all three chains in the most proximal region of the long arm and join the xcex2 and xcex3 chains in the most distal region of the long arm.
A model of laminin receptor-facilitated self-assembly, based on studies conducted with cultured skeletal myotubes and Schwann cells, predicts that laminins bind to their receptors, which freely diffuse in a fluidic membrane, when ligand-free. Receptor engagement forces these receptors into a high local two-dimensional concentration, facilitating their mass-action driven assembly into ordered surface polymers. In this process, the engaged receptors are also reorganized, accompanied by cytoskeletal rearrangements. (Colognato, J. Cell Biol. 145:619-631 (1999)) This reorganization activates the receptors, causing signal transduction with the alteration of cell expression, shape and/or behavior.
One class of laminin receptors are the integrins, which are cell surface receptors that mediate many cell-matrix and cell-cell interactions. Integrins are heterodimers, consisting of an xcex1 and xcex2 subunit. 16 xcex1- and 8 xcex2-subunits are known, and at least 22 combinations of xcex1 and xcex2 subunits have been identified to date. Some integrins have only one or a few known ligands, whereas others appear to be very promiscuous. Binding to integrins is generally of low affinity, and is dependent on divalent cations. Integrins, activated through binding to their ligands, transduce signals via kinase activation cascades, such as focal adhesion and mitogen-activated kinases. Several different integrins bind different laminin isoforms more or less specifically. (Aumailley et al., In The Laminins, Timpl and Ekblom, eds., Harwood Academic Publishers, Amsterdam. pp. 127-158 (1996))
Laminin-5, also referred to as kalinin, nicein, and epiligrin, plays a key role in modulating the behavior and activity of cells and tissues of epithelial origin, and is expected to have broad uses in clinical settings where increased epithelial attachment and hemidesmosome assembly are required. (Takeda et al., J. Invest. Dermatol. 1999 113(1):38-42) Laminin-5 is a principal adhesive ligand in the epidermal basal lamina, and has been shown to promote the attachment of keratinocytes and epithelial cells to the basal lamina and underlying dermis, and also promotes hemidesmosome formation. (Burgeson et al. U.S. Pat. No. 5,770,562; Quaranta and Hormia, U.S. Pat. No. 5,422,264; Jones, U.S. Pat. No. 5,541,106; Quaranta and Hornia, U.S. Pat. No. 5,658,789; Hormia et al., J. Invest. Dermatol. 1995 October 105(4):557-561).
Laminin 5 is also thought to be necessary for the healing of epithelial tissue wounds. (Goldfinger et al., J. Cell Sci. 1999; 112(Pt. 16):2615-2629) Pretreatment of human keratinocyte sheets for grafting with laminin 5 improves grafting efficiency. (Takeda et al., J. Invest. Dermatol. 1999 July; 113(1):38-42). The addition of laminin-5 accelerates the formation of a basement membrane in a skin equivalent model (Tsunenaga et al., Matrix Biol. 17(8-9):603-613, 1998). Laminin-5 also promotes epithelial cell attachment to a wide variety of biomaterials, including polymers, hydroxyapatite, and metals. (Jones et al., U.S. Pat. No. 5,585,267; El Ghannam et al., J. Biomed. Mater. Res. 1998 July; 41(1):30-40).
Laminin 5 has further been demonstrated to promote the following:
1. Epithelial cell adhesion to the internal basal lamina of teeth (Mullen et al., J. Periodontal. Res. 1999 January 34(1):16-24; Hormia et al., J. Dent. Res. 1998 July; 77(7):1479-1485). and anchorage of ameloblasts (ie: enamel-producing cells) to the enamel matrix. (Yoshiba et al., Cell Tissue Res. 1998 April; 292(1):143-149).
2. Corneal epithelial cell adhesion. (Qin and Kurpakus, Exp. Eye Res. 1998 May 66(5):569-579).
3. Intestinal epithelial restitution. (Lotz et al., Am. J. Pathol. 1997 February;150(2):747-760).
4. In vitro expansion of epithelial cells by providing an efficient adhesion substrate for primary cell cultures, thus providing the basis for a wide range of new cell therapy applications. (Gonzales et al., Mol. Biol. Cell. 1999 February; 10(2):259-270; Baker et al., Exp. Cell Res. Nov. 1, 1996; 228(2)262-270).
5. Proliferation of pancreatic beta islet cells (Todorov et al., Transplant. Proc. 1998 March; 30(2): 455; Quaranta and Jones, U.S. Pat. No. 5,510,263; Halberstadt et al, U.S. Pat. No. 5,681,587; Halberstadt et al., U.S. Pat. No. 5,672,361), and T cells (Vivinus-Nebot et al., J. Cell Biol. Feb. 8, 1999; 144(3):563-574).
Thus, laminin 5 has broad uses in clinical settings where increased epithelial attachment and hemidesmosome assembly are required. Laminin 5 can be used to promote wound healing and tissue regeneration. The application of exogenous laminin 5 has broad application for the accelerated healing of skin disorders, such as diabetic foot ulcers, venous ulcers, pressure sores, skin surgery, bums, and acute wounds. Exogenous laminin 5 may be used to directly treat a wound surface, or may be applied to a variety of medical devices and dermal grafts for skin, corneal, gastrointestinal, and periodontal epithelial wound healing. The use of laminin 5 provides enhanced biocompatibility of the device or graft, which leads to improved tissue integration and remodeling, reduced immune response, and reduced likelihood of infection. Laminin 5 is also useful for the ex vivo and in vitro proliferation of various cell types, including but not limited to epithelial cells, pancreatic beta islet cells, and T cells, and tissue equivalents. Thus, a source of laminin 5 for tissue culture media or a media supplement, as well as cell growth substrates coated with laminin 5, would be particularly useful for the cultivation of these and other cell types.
A good source and purification procedure for laminin-5 is needed to provide material for the development of the therapeutic and research applications mentioned above. Some cell lines secrete laminin-5, and procedures have been developed to purify laminin-5 from the processed cells and cell media. However, these methods are time consuming and capable of producing only small amounts of laminin 5. (Rouselle et al., J. Biol. Chem. 1995 270(23):13766-13770; Cheng et al., J. Biol. Chem. 1997, 272(50):31525-31532).
A preferred method of production, however, is the use of recombinant DNA technology to engineer a cell line of choice to produce recombinant laminin-5. A recombinant-based method of laminin-5 production has several advantages over purification from tissue or isolation from cell lines in culture:
1. The recombinant produced protein is free of pathogens. While this is also true for endogenous cell culture produced protein, protein derived from human tissue carries a risk for contamination by HIV, hepatitis, and other infectious agents.
2. Expression levels of the protein, and hence yields, can be improved through the use of genetically engineered genes/vectors that enhance the production of the encoded protein.
3. It is possible to engineer additional peptide sequences to the protein chain that provides a binding site for a commercially viable affinity purification procedure.
4. The method can provide for the modification of protein structure/function through the addition, substitution, elimination, and/or other modifications of protein domain structures. For example, it may be desirable to introduce an integrin binding site (e.g. RGD), switch integrin recognition sites, or engineer in a stable binding site to a synthetic substrate. Thus, the creation of expression vectors that express laminin chains generates enormous flexibility for future uses and creates a basis for creating second generation xe2x80x9cdesignerxe2x80x9d laminins.
Previous studies have produced cells transfected with one or two of the laminin 5 chain-encoding DNA sequences, but none have produced recombinant heterotrimeric laminin 5, nor have they produced cell lines that secrete recombinant heterotrimeric laminin 5. (Gagnoux-Palacios et al., J. Biol. Chem. 271:18437-18444 (1996); Matsui et al., J. Biol. Chem. 270:23496-23503 (1995)).
Thus, there exists a need in the art for recombinant heterotrimeric laminin 5 protein, methods for making recombinant laminin 5, and methods of using recombinant laminin 5 for wound healing and tissue regeneration, for use on a variety of medical devices and dermal grafts for skin, comeal, gastrointestinal, and periodontal epithelial wound healing, for the ex vivo and in vitro proliferation of various cell types, and for tissue culture media, media supplements, and as a component of cell growth substrates.
The present invention fulfills the need in the art for recombinant laminin 5 protein, methods for making recombinant laminin 5, and methods of using recombinant laminin 5 for the treatment of burns, for use on a variety of medical devices and dermal grafts for skin, corneal, gastrointestinal, and periodontal epithelial wound healing, for the ex vivo and in vitro proliferation of various cell types, and for tissue culture media, media supplements, and as a component of cell growth substrates.
In one aspect, the present invention provides cells that have been transfected with nucleic acid sequences encoding laminin xcex13, xcex23 and xcex32 chains, wherein the cells express the individual chains, which assemble into heterotrimeric recombinant laminin-5 (hereinafter referred to as xe2x80x9cr-laminin 5xe2x80x9d). r-laminin 5, or processed forms thereof, are secreted by the cell.
In another aspect, the present invention provides r-laminin 5, and methods for producing substantially purified r-laminin 5, or processed forms thereof.
In a further aspect, the present invention provides pharmaceutical compositions, comprising r-laminin 5, or processed forms thereof, together with a pharmaceutically acceptable carrier. Such pharmaceutical compositions can optionally be provided with other compounds with wound healing and tissue regeneration utility, such as other extracellular matrix components.
In further aspect, the present invention provides methods and kits for using r-laminin 5 to:
a. accelerate wound healing and tissue regeneration;
b. enhance the performance of skin grafts;
c. improve the attachment of gum tissue to the tooth surface;
d. improve the biocompatibility of medical devices; and
e. accelerate cell proliferation,
xe2x80x83by providing an amount effective of r-laminin 5 for the various methods. The invention also provides methods and kits for using laminin 5 to regulate angiogenesis. The kits comprise an amount of laminin 5 or r-laminin 5 effective for the desired effect, and instructions for the use thereof.
In a further aspect, the present invention provides improved medical devices and grafts, wherein the improvement comprises coating the devices or grafts with an amount effective of r-laminin 5 or the pharmaceutical compositions of the invention for the desired application.
In a further aspect, the invention provides improved cell culture devices for the proliferation of cells in culture, by providing an amount effective of r-laminin 5 for the attachment of cells to a cell culture device for the attachment and subsequent proliferation, differentiation, or maintenance of the cells.
In another aspect, the invention provides a cell culture growth supplement, comprising r-laminin 5. In another aspect, the invention provides an improved cell culture growth media, wherein the improvement comprises the addition of r-laminin 5.